Chemical analysis of biological samples is constrained by sample size. Withdrawing a few milliliters of blood from an adult may have little effect, but repeating this procedure every hour or even withdrawing this amount once from an infant can significantly alter the health of the subject. For these reasons, a miniaturized blood analysis system would be useful. Furthermore, while many sophisticated tests that have great importance for critical care can be performed in major hospital laboratories, a substantial impact could be made on the practice of emergency medicine if some key tests could be performed on the patient at the site of injury. For some assays it is vital to make measurements in the absence of red blood cells, so some form of separation of cells from plasma is required.
Diffusion is a process which can easily be neglected at large scales, but rapidly becomes important at the microscale. The average time t for a molecule to diffuse across a distance d is 2t=d.sup.2 /D where D is the diffusion coefficient of the molecule. For a protein or other large molecule, diffusion is relatively slow at the macroscale (e.g. hemoglobin with D equal to 7.times.10.sup.-7 cm.sup.2 /s in water at room temperature takes about 10.sup.6 seconds (ten days) to diffuse across a one centimeter pipe, but about one second to diffuse across a 10 .mu.m channel).
Using tools developed by the semiconductor industry to miniaturize electronics, it is possible to fabricate intricate fluid systems with channel sizes as small as a micron. These devices can be mass-produced inexpensively and are expected to soon be in widespread use for simple analytical tests. See, e.g., Ramsey, J. M. et al. (1995), "Microfabricated chemical measurement systems," Nature Medicine 1:1093-1096; and Harrison, D. J. et al (1993), "Micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip," Science 261:895-897.
Miniaturization of analytic instruments is not a simple matter of reducing their size. At small scales different effects become important, rendering some processes inefficient and others useless. It is difficult to replicate smaller versions of some devices because of material or process limitations. For these reasons it is necessary to develop new methods for performing common laboratory tasks on the microscale.
Devices made by micromachining planar substrates have been made and used for chemical separation, analysis, and sensing. See, e.g., Manz, A. et al. (1994), "Electroosmotic pumping and electrophoretic separations for miniaturized chemical analysis system," J. Micromech. Microeng. 4:257-265.
Field flow fractionation devices involve particle size separation using a single inlet stream. See, e.g. Giddings, J. C., U.S. Pat. No. 3,449,938, Jun. 17, 1969, "Method for Separating and Detecting Fluid Materials"; Giddings, J. C., U.S. Pat. No. 4,147,621, Apr. 3, 1979, "Method and Apparatus for Flow Field-Flow Fractionation"; Giddings, J. C., U.S. Pat. No. 4,214,981, Jul. 29, 1980, "Steric Field-Flow Fractionation"; Giddings, J. C. et al., U.S. Pat. No. 4,250,026, Feb. 10, 1981, "Continuous Steric FFF Device for The Size Separation of Particles"; Giddings, J. C. et al. (1983), "Outlet Stream Splitting for Sample Concentration in Field-Flow Fractionation," Separation Science and Technology 18:293-306; Giddings, J. C. (1985), "Optimized Field-Flow Fractionation System Based on Dual Stream Splitters," Anal. Chem. 57:945-947; Giddings, J. C., U.S. Pat. No. 4,830,756, May 16, 1989, "High Speed Separation of Ultra-High Molecular Weight Polymers by Hyperlayer Field-Flow Fractionation"; Giddings, J. C., U.S. Pat. No. 4,141,651, Aug. 25, 1992, "Pinched Channel Inlet System for Reduced Relaxation Effects and Stopless Flow Injection in Field-Flow Fractionation"; Giddings, J. C., U.S. Pat. No. 5,156,039, Oct. 20, 1992, "Procedure for Determining the Size and Size Distribution of Particles Using Sedimentation Field-Flow Fractionation"; Giddings, J. C., U.S. Pat. No. 5,193,688, Mar. 16, 1993, "Method and Apparatus for Hydrodynamic Relaxation and Sample Concentration in Field-Flow Fraction Using Permeable Wall Elements"; Caldwell, K. D. et al., U.S. Pat. No. 5,240,618, Aug. 31, 1993, "Electrical Field-Flow Fractionation Using Redox Couple Added to Carrier Fluid"; Giddings, J. C. (1993), "Field-Flow Fractionation: Analysis of Macromolecular, Colloidal and Particulate Materials," Science 260:1456-1465; Wada, Y. et al., U.S. Pat. No. 5,465,849, Nov. 14, 1995, "Column and Method for Separating Particles in Accordance with Their Magnetic Susceptibility"; Yue, V. et al. (1994), "Miniature Field-Flow Fractionation Systems for Analysis of Blood Cells," Clin. Chem. 40:1810-1814; Afromowitz, M. A. and Samaras, J. E. (1989), "Pinch Field Flow Fractionation Using Flow Injection Techniques," Separation Science and Technology 24(5 and 6):325-339.
Thin-channel split flow fractionation (SPLITT) technology also provides particle separation in a separation cell having a thin channel. A field force is exerted in a direction perpendicular to the flow direction. Particles diffuse or are otherwise transported from a particle-containing stream across a transport stream to a particle-free stream. The device for operating the process is generally fabricated from glass plates with teflon sheets used as spacers to form the channels. The channel depth can therefore be no smaller than the spacers, which are generally about 100 to 120 .mu.m thick. See, e.g., Giddings, J. C., U.S. Pat. No. 4,737,268, Apr. 12, 1988, "Thin Channel Split Flow Continuous Equilibrium Process and Apparatus for Particle Fractionation"; Giddings, J. C., U.S. Pat. No. 4,894,146, Jan. 16, 1990, "Thin Channel Split Flow Process and Apparatus for Particle Fractionation"; Giddings, J. C., U.S. Pat. No. 5,093,426, Aug. 13, 1991, "Process for Continuous Particle and Polymer Separation in Split-Flow Thin Cells Using Flow-Dependent Lift Forces"; Williams, P. S. et al. (1992), "Continuous SPLITT Fractionation Based on a Diffusion Mechanism," Ind. Eng. Chem. Res. 31:2172-2181; and Levin, S. and Tawil, G. (1993), "Analytical SPLITT Fractionation in the Diffusion Mode Operating as a Dialysis-like System Devoid of Membrane. Application to Drug-Carrying Liposomes," Anal. Chem. 65:2254-2261.
The object of this invention is to provide a microfabricated extraction system utilizing differential transport principles in which an analyte can be extracted, detected and quantified.
The advantages, as disclosed herein, of diffusion separation devices on the microscale, e.g., having channel depths no greater than about 100 .mu.m, do not appear to have been recognized in the prior art. See, e.g., Kittilsand, G. and Stemme, G. (1990), Sensors and Actuators A21-A23:904-907, and Wilding, P. et al. (1994), J. Clin. Chem. 40:43-47.
All publications, patents and patent applications referred to herein are hereby incorporated by reference.